Proteins, acting directly or through their enzymatic functions, contribute in major proportion to many diseases in animals and humans. Classical therapeutic methods have generally focused on modulating protein function with exogenous compounds that interact directly with proteins, with the goal of moderating their disease-causing or disease-potentiating functions. Recently, however, attempts have been made to affect the production of certain proteins by modulating the activity of molecules that direct protein synthesis, such as intracellular RNA. By interfering with the production of specific proteins, it has been hoped to effect therapeutic results with maximal desired effect and minimal side effects.
One method for inhibiting specific gene expression involves using oligonucleotides or oligonucleotide analogs as “antisense” agents. Antisense technology involves directing oligonucleotides, or analogs thereof, to a specific, target messenger RNA (mRNA) sequence. The interaction of exogenous “antisense” molecules and endogenous mRNA modulates transcription by a variety of pathways. Such pathways include transcription arrest, RNAse H recruitment, and RNAi (e.g. siRNA). Antisense technology permits modulation of specific protein activity in a relatively predictable manner.
In fact, antisense oligonucleotides and oligonucleotide analogs are now accepted as therapeutic agents that hold great promise for therapeutic and diagnostic methods. Accordingly, it has become desirable to produce oligonucleotides and their analogs in relatively large quantities. In some applications, it is necessary to produce large numbers of small batches of diverse oligonucleotides or their analogs for screening purposes. In other cases, for example in the production of therapeutic quantities of oligonucleotides and their analogs, it is necessary to make large batches of the same oligonucleotide, or analog thereof.
Three principal methods have been used for the synthesis of oligonucleotides. The phosphotriester method, as described by Reese, Tetrahedron 1978, 34, 3143; the phosphoramidite method, as described by Beaucage, in Methods in Molecular Biology: Protocols for Oligonucleotides and Analogs; Agrawal, ed.; Humana Press: Totowa, 1993, Vol. 20, 33-61; and the H-phosphonate method, as described by Froehler in Methods in Molecular Biology: Protocols for Oligonucleotides and Analogs Agrawal, ed.; Humana Press: Totowa, 1993, Vol. 20, 63-80. Of these three methods, the phosphoramidite method has become a defacto standard in the industry.
A typical oligonucleotide synthesis using phosphoramidite chemistry (i.e. the amidite methodology) is set forth below. First, a primer support is provided in a standard synthesizer column. The primer support is typically a solid support (supt) having a linker (link) covalently bonded thereto. It is common to purchase the primer support with a first 5′-protected nucleoside bonded thereto.
wherein bg is a 5′-blocking group, Bx is a nucleobase, R2 is H, OH, OH protected with a removable protecting group, or a 2′-substituent, such as 2′-deoxy-2′-methoxyethoxy (2′-MOE), link is the covalent linking group, which joins the nucleoside to the support, supt.    (A) The 5′-blocking group bg (e.g. 4,4′-dimethoxytrityl) is first removed (e.g. by exposing the 5′-blocked primer-support bound nucleoside to an acid, thereby producing a support-bound nucleoside of the formula:
wherein supt is the solid support, link is the linking group, Bx is a nucleobase, R2 is H, OH, OH protected with a removable protecting group, or a 2′-substituent.    (B) The column is then washed with acetonitrile, which acts to both “push” the regent (acid) onto the column, and to wash unreacted reagent and the removed 5′-blocking group (e.g. trityl alcohol) from the column.    (C) The primer support is then reacted with a phosphitylation reagent (amidite), which is dissolved in acetonitrile, the amidite having the formula:
wherein bg is a 5′-blocking group, Ig is a leaving group, G1 is O or S, pg is a phosphorus protecting group, and R2 and Bx have, independent of the analogous variables on the primer support, the same definitions as previously defined.
The product of this reaction is the support-bound phosphite dimer:
wherein each of the variables bg, G1, pg, R2 and Bx is independently defined above, link is the linker and supt is the support, as defined above.    (D) The support-bound dimer is then typically washed with acetonitrile.    (E) The support-bound dimer is then typically reacted with an oxidizing agent, such as a thiating agent (e.g. phenylacetyl disulfide), in acetonitrile, to form a support-bound phosphate triester:
wherein each of G and G1 is, independently, O or S and the other variables are defined herein.    (F) The column is then washed again with acetonitrile.    (G) A capping reagent in acetonitrile is then added to the column, thereby capping unreacted nucleoside.    (H) The support-bound phosphate triester is then typically washed with acetonitrile.Steps (A)-(H) are then repeated, if inecessary, a sufficient number of times (n−1) to prepare a support-bound, blocked oligonucleotide having the formula:
wherein n is a positive integer (typically about 7 to about 79).
The phosphorus protecting groups pg are then typically removed from the oligomer to produce a support-bound oligomer having the formula:
which, after washing with a suitable solvent wash, such as acetonitrile, is typically cleaved from the solid support, purified, 5′-deblocked, and further processed to produce an oligomer of the formula:

The foregoing methodology has historically proven effective in the production of small- to medium-scale quantities of oligonucleotide. In fact, heretofore it has been believed that acetonitrile is the best solvent for use in oligonucleotide synthesis, including dissolution and introduction of reagents to the column, as well as for column washing steps between reagent addition steps. It has been believed, in fact, that the polarity, viscosity and other characteristics of acetonitrile made it the solvent of choice for solid phase oligonucleotide synthesis. However, acetonitrile is a relatively expensive solvent. If acetonitrile could be replaced with a less-costly solvent, it could potentially produce extensive cost savings, especially as the scale of oligonucleotide synthesis increases. Nevertheless, the long-held belief in the art was that acetonitrile could not be replaced as a solvent without sacrificing oligonucleotide purity or yield, either one of which would be unacceptable in view of the high cost of raw materials such as amidites.
There is therefore a need for a substitute for acetonitrile as a solvent in oligonucleotide synthesis.
There is also a need for a substitute solvent wash for oligonucleotide synthesis.
There is also a need for a reagent push other than acetonitrile for use in oligonucleotide synthesis.
There is also a need for an oligonucleotide synthetic method using an alternative solvent wash that supports production of oligonucleotides in purity at least as good as acetonitrile.
There is also a need for an oligonucleotide synthetic method using an alternative solvent wash that supports production of oligonucleotides in yields at least as good as those supported by acetonitrile.
There is also a need for an oligonucleotide synthetic method using an alternative solvent wash that is less expensive than acetonitrile.